A structural analysis of DNA binding by myelin transcription factor 1 double zinc fingers.

EXPERIMENTAL PROCEDURES Subcloning, Expression, and Purification of MyT1 Constructs The original plasmid encoding mouse 6-ZF myelin transcription factor 1 (mMyT1) was a gift of Dr Lynn Hudson (National Institutes of Health). Both F4F5 and F5F6 constructs of MyT1 were cloned from the original plasmid (residues 18–904), and mutants were constructed using either overlap extension PCR or site-directed mutagenesis. All constructs were cloned into the pGEX-6P vector and overexpressed as GST fusions at 37 °C under standard conditions; isotopically labeled proteins were overexpressed using the protocol described previously (15). Proteins were purified using GSH affinity chromatography, HRV-3C cleavage, and gel filtration (Superdex-75 in SPR buffer: 50 mM NaCl, 10 mM HEPES, 1 mM DTT, pH 7.2). Protein concentrations were determined by absorbance at 215, 225, and 280 nm (16). Fractions were stored in the presence of protease inhibitors at −20 °C until required. β-RARE DNA and Mutant Oligonucleotides Single-stranded β-RARE DNA (5′-ACCGAAAGTTCAC and 5′-GTGAACTTTCGGT), mutant oligonucleotides and biotinylated DNA for SPR experiments were obtained from Sigma-Aldrich, annealed in SPR buffer without DTT (heated to 95 °C for 5 min and then cooled to room temperature over the course of 1–2 h), and purified using gel filtration (Superdex-75). Concentrations were calculated from absorbance at 260 nm. Surface Plasmon Resonance All experiments were performed on a Biacore 3000 system (Biacore AB) at flow rates of 20 μl/min in SPR buffer to which was added 0.01% polysorbate 20 (P20) detergent. Biotinylated DNA (∼10–100 nM) was immobilized on streptavidin-coated Biacore SA chips (50–100 resonance units). MyT1 and MyT1 mutants (0.2–10 μM) were injected in SPR buffer, and binding was monitored. The system was washed with 1 M NaCl (1 min) after each experiment. For kinetics studies, the Biacore BiaEvaluation software was utilized to calculate affinity constants using global fitting algorithms. In the competition experiments, F5F6 (5 μM) was added to prebound DNA in the presence of 5 molar eq of competitor DNA oligonucleotides. NMR Spectroscopy F4F5 or F5F6 (unlabeled, 15N-labeled, or 15N/13C-labeled) were exchanged into NMR buffer (50 mM NaCl, 10 mM phosphate, 1 mM DTT, pH 7.2) with 1 mM DTT and concentrated in Microsep 3K cutoff filters to 200–1000 μM. Resonance assignments were made from standard triple-resonance experiments that were acquired at 25 °C on Bruker Avance 600 and 800 NMR spectrometers equipped with cryoprobes. 15N HSQC titrations as well as two-dimensional NOESY experiments of proteins with β-RARE DNA were carried out in NMR buffer at 25 °C. Chemical shift changes were calculated as a weighted average of HN, N, and Cα changes, using a previously reported equation (17, 18). Assignments of the DNA alone were obtained from our previous study (14). One-bond HN residual dipolar couplings (RDCs) were recorded for the F4F5-DNA complex in NMR buffer containing 22.2 mg/liter Pf1 phage (ASLA Biotech), using the in-phase/anti-phase pulse sequence (19). Alignment was assessed by measuring the D2O splitting (19 Hz). The program PALES (20) was used for the calculation of the magnitude and orientation of the sterically induced alignment tensor (see below for details). NMR data were processed using Topspin (Bruker, Karlsruhe) and analyzed with SPARKY 3 (37). HADDOCK Docking F4F5 was docked to the DNA using the program HADDOCK 1.3 (21–23). The starting structure for the DNA was a B-form model of the double helix DNA fragment (5′-ACCGAAAGTTCAC) constructed with the Nucleic Acid Builder package (24). Based on our NMR data (see Figs. 2 and 3), a starting structure of F4F5 was made in silico by fusing two individual ZF domains and the native linker sequence (see Fig. 1) together using the calculated NMR structure of F5 (14) (Protein Data Bank (PDB) ID 2JYD) as a template. A total of 10 different starting orientations between F4F5 and the DNA were chosen as starting structures for the docking. Sequences shown to be disordered in our previous NMR analysis, namely residues 799–800 (N-terminal) and 872–873 (C-terminal), were defined as fully flexible during the calculations, as was the internal linker (828–845) (14). Ambiguous interaction restraints for both the protein and the DNA were chosen based both on our NMR data from Figs. 2 and 3 and on solvent accessibility (>30%, determined by the program MOLMOL) and were fixed at 2 Å. For the DNA fragment, ambiguous interaction restraints were defined solely from the unique base atoms of bases Ade6, Ade7, Thy20, and Thy21, whereas for F5, DNA bases Thy9, Thy10, Ade17, and Ade18 were selected. For the protein, restraints between unique side-chain atoms of F4 (residues His-812, Tyr-817, Ser-819, Arg-821, Ser-822, Leu-823, Ser-824) as well as corresponding residues in F5 (His-856, Tyr-861, Ser-863, Arg-865, Ser-866, Leu-867, Ser-868) were chosen. A total of 48 ambiguous interaction restraints resulted from these definitions and were used as input into HADDOCK for all 10 different F4F5-DNA starting configurations. Additional restraints to maintain base planarity and Watson-Crick bonds for the DNA, intramolecular noncrystallographic symmetry restraints between F4F5 and DNA (F4+Ade6/7 = F5+Ade17/18 and F4+Thy20/21 = F5+Thy9/10), and zinc-coordinating restraints for F4F5 were introduced. During the rigid body energy minimization, 1000 structures were calculated, and the 200 best solutions based on the intermolecular energy were used for the semiflexible, simulated annealing. 10 different runs were carried out with the 10 F4F5-DNA starting orientations, respectively. The best 10 structures of each run were part of the lowest energy cluster (cut-off of 0.5 Å root mean square deviation (RMSD) based on the pairwise backbone RMSD matrix). The 10 best structures from run I were subjected to a second round of semiflexible annealing following the inclusion of 43 HN RDCs as additional direct restraints (using the SANI statement); axial and rhombic components of the alignment tensor (Da and Dr) were calculated using the 10 run I structures and the software PALES (20). The alignment tensor was then recalculated based on the resulting best 10 of a total of 200 calculated structures (lowest SANI energies), and HADDOCK was run again (see above) using these new values. After this protocol, the final 10 structures were not significantly different from the ones calculated without the RDCs (RMSD over all atoms of the lowest energy structure = 0.3 Å). These structures were analyzed using standard HADDOCK protocols. PRE Measurements To attach a paramagnetic moiety to the RARE oligonucleotide, lyophilized modified RARE DNA containing a phosphorothioate linkage at Thy15 (which is located next to the DNA-binding site) was resuspended in 100 mM phosphate buffer (pH 7) to a concentration of ∼400 μM. This solution was then used to dissolve the complementary strand, and the DNA was annealed by heating to 95 °C for 5 min and then cooling to room temperature over a period of 1 h. A 100 molar excess of 3-(2-iodoacetamido-)proxyl radical (in 100% ethanol) was added to the annealed oligonucleotide and incubated in the dark for 20 h under shaking. To remove excess single-stranded DNA and unreacted 3-(2-iodoacetamido-)proxyl, the reaction mixture was subjected to size exclusion chromatography using NMR buffer. The progress of the reaction was monitored using UV spectroscopy and mass spectroscopy. The final product (containing about 50% labeled and 50% unlabeled double-stranded DNA) was used to carry out a semiquantitative paramagnetic resonance enhancement (PRE) analysis. To acquire PRE data, two-time point HSQC experiments as described in Ref. 25 were performed (Ta = 0 ms; Tb = 14 ms) on a 1 mM F5-DNA complex. Spectra were first recorded for the paramagnetic (oxidized) sample and then for the diamagnetic (reduced) sample following the addition of a 50 molar excess of sodium dithionate. After normalizing all four 15N HSQC spectra, peak intensities were obtained for the assigned residues and PRE rates (25) were calculated. Distances shown in Fig. 8B are calculated from the phosphate of Thy15 to the HN backbone atom of the corresponding residue.Background: Myelin transcription factor 1 (MyT1) contains seven similar zinc finger domains that bind DNA specifically. Results: A three-dimensional structural model explains how a double zinc finger unit is able to recognize DNA. Conclusion: DNA-binding residues are conserved among all MyT1 zinc fingers, suggesting an identical DNA binding mode. Significance: Determination of the molecular details of DNA interaction will be crucial in understanding MyT1 function. Abstract: Myelin transcription factor 1 (MyT1/NZF2), a member of the neural zinc-finger (NZF) protein family, is a transcription factor that plays a central role in the developing central nervous system. It has also recently been shown that, in combination with two other transcription factors, the highly similar paralog MyT1L is able to direct the differentiation of murine and human stem cells into functional neurons. MyT1 contains seven zinc fingers (ZFs) that are highly conserved throughout the protein and throughout the NZF family. We recently presented a model for the interaction of the fifth ZF of MyT1 with a DNA sequence derived from the promoter of the retinoic acid receptor (RARE) gene. Here, we have used NMR spectroscopy, in combination with surface plasmon resonance and data-driven molecular docking, to delineate the mechanism of DNA binding for double ZF polypeptides derived from MyT1. Our data indicate that a two-ZF unit interacts with the major groove of the entire RARE motif and that both fingers bind in an identical manner and with overall two-fold rotational symmetry, consistent with the palindromic nature of the target DNA. Several key residues located in one of the irregular loops of the ZFs are utilized to achieve specific binding. Analysis of the human and mouse genomes based on our structural data reveals three putative MyT1 target genes involved in neuronal development. This work was supported by the National Health and Medical Council of Australia (NHMRC) and the Australian Research Council (ARC).